1. Field of the Invention
This invention relates generally to the detection of nucleic acid sequences by polymerase chain reaction (PCR). More particularly, this invention relates to a process for efficiently producing single-stranded PCR products in an amount proportional to the amount of a target nucleic acid sequence present in a sample being analyzed.
2. Description of the Art
PCR is a commonly used technique for the detection of target nucleic acid sequences in a sample due to its exquisite specificity and sensitivity. Specificity is achieved by the use of two oligonucleotide primers whose sequences are complementary to sequences that define a deoxyribonucleic acid (DNA) segment containing the target sequence. Following heat denaturation of the target DNA, these primers anneal to their complementary sequences and are extended using a DNA polymerase enzyme and nucleotides. This can be thought of as a symmetrical process: the primers are selected to hybridize to opposite strands of the target sequence in an orientation such that DNA synthesis by the polymerase fills in the region between the primers. Thus, a double-stranded DNA replica, or PCR product, of the original target nucleic acid sequence is produced.
Repetitions of this cycle of denaturation, primer annealing, and extension results in the exponential accumulation of PCR products. This exponential amplification provides the sensitivity of PCR, allowing the detection of extremely small amounts of nucleic acid molecules containing the target sequence, i.e. a single DNA molecule has been amplified and detected. Target sequences in ribonucleic acid (RNA) can also be amplified and detected by performing PCR on complementary DNA (cDNA) produced from an RNA template.
PCR products are generally analyzed by gel electrophoresis or by hybridization with a nucleic acid probe. In many instances, detection of PCR products with nucleic acid probes is the preferred analytical method because it is faster and more economical than gel electrophoresis.
However, analysis of PCR products with nucleic acid probes is a multistep process. First, the double-stranded PCR products are denatured to generate a mixture of single-stranded PCR products. Next, a nucleic acid probe hybridizes to the strand of the mixture that has a sequence complementary to that of the probe to form a probe-PCR product hybrid molecule. Finally, the probe-PCR hybrid is detected.
This process is inefficient because denatured PCR products rapidly renature. That is, the two strands of denatured DNA products quickly reassociate with each other and reform the double-stranded PCR products, thereby excluding the nucleic acid probe. This renaturation problem can be avoided by modifying PCR such that single-stranded DNA of a chosen strand is the major PCR product. A prior art process for producing single-stranded PCR product is asymmetric PCR. (See, e.g., U. B. Gyllensten and H. A. Erlich, "Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus." Proc. Natl. Acad. Sci. USA, vol. 85, pp. 7652-7656 (1988), herein incorporated by reference.)
Asymmetric PCR uses an unequal, or asymmetric, concentration of the two amplification primers. For example, typical primer ratios for asymmetric PCR are 50:1 to 100:1. During the initial 15 to 25 cycles of asymmetric PCR, most of the product generated is double-stranded and accumulates exponentially. However, as the low-concentration primer becomes depleted, further cycles generate an excess of one strand, i.e. , the strand that is complementary to the limiting primer. This single-stranded DNA accumulates linearly resulting in a PCR product mixture that contains both double-stranded DNA and single-stranded DNA. Consequently, renaturation of the double-stranded PCR product is an insignificant factor, since detection of the single-stranded PCR product with a nucleic acid probe is highly efficient.
Besides detecting the presence of a DNA or RNA sequence in a sample, some determinations also require quantifying the number of molecules having the target sequence that are initially present in a sample. For example, quantitative information is required for the analysis of the induction of mRNA in response to exogenous stimuli, gene amplification in tumors and the progress of some viral infections. Quantitative determinations using symmetric PCR are well known in the art. In brief, the quantity of DNA or cDNA in a sample is determined by comparing the amounts of PCR products that result from co-amplification of a target sequence and an added internal standard of known concentration that is amplified by the same primers. (See, e.g., P. D. Seibert and J. W. Larrick, Nature, vol. 359, pp. 557-558 (1992), herein incorporated by reference. ) However, as discussed above, symmetric PCR is not a satisfactory method for amplifying target sequences for detection by nucleic acid probe hybridization due to the need for denaturation of the double-stranded PCR products and the complications arising from PCR product reannealing.
Although asymmetric PCR overcomes these deficiencies of symmetric PCR in providing efficient detection, asymmetric PCR is not suitable for quantitative determinations because the amount of single-stranded PCR product generated after amplification is primarily determined by the amount of limiting primer added to the reaction, not by the amount of target DNA or RNA originally present in the sample. For example, increasing quantities of target in the sample results only in fewer cycles being required to exhaust the limiting primer. Thus, quantitation of target sequences amplified by asymmetric PCR is only possible within a 1000-fold range of initial target sequence concentration and even within that range, a 10-fold increase in target produces only a 2-fold increase in detection.
From the foregoing, it will be readily apparent to those skilled in the art that for applications requiring both efficient detection and accurate quantitation of target sequences by nucleic probe hybridization, neither of the :prior art PCR processes discussed above is satisfactory. Therefore, those skilled in the art would appreciate the usefulness of a process that generates a large number of single-stranded copies of a target sequence in an amount that is proportional to the number of target nucleic acid molecules originally present in a sample.